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| United States Patent |
5,629,153
|
|
Urdea
|
May 13, 1997
|
Use of DNA-dependent RNA polymerase transcripts as reporter molecules
for signal amplification in nucleic acid hybridization assays
Abstract
A polydeoxynucleotide construct is disclosed for use, in conjunction with a
DNA-dependent RNA polymerase, as a signal amplifier in nucleic acid
hybridization assays. The construct contains a recognition sequence for a
target oligonucleotide, a promoter sequence for a DNA-dependent RNA
polymerase, and a polymerase template. A method of use for this construct
in hybridization assays is also disclosed. The method involves formation
of a hybridization complex comprising the construct and the target
sequence; addition of a polymerase which is specific for the promoter in
the construct; and quantification of the resulting RNA transcripts.
| Inventors:
|
Urdea; Michael S. (Alamo, CA)
|
| Assignee:
|
Chiron Corporation (Emeryville, CA)
|
| Appl. No.:
|
207901 |
| Filed:
|
March 8, 1994 |
| Current U.S. Class: |
435/6; 435/91.2; 435/91.21; 536/24.3; 536/24.32 |
| Intern'l Class: |
C12Q 001/68; C12P 019/34; C07H 021/04 |
| Field of Search: |
435/6,91.2,91.21
536/24.3,24.33,24.2
935/77,78,79
|
References Cited
U.S. Patent Documents
| 4868105 | Sep., 1989 | Urdea et al. | 435/6.
|
| 5112734 | May., 1992 | Kramer et al. | 435/6.
|
| 5124246 | Jun., 1992 | Urdea et al. | 435/6.
|
| 5356774 | Oct., 1994 | Axelrod et al. | 435/6.
|
| Foreign Patent Documents |
| 0204510 | Dec., 1986 | EP | .
|
| 0317077 | May., 1989 | EP | .
|
| 0346594 | Dec., 1989 | EP | .
|
| 0369775A3 | May., 1990 | EP.
| |
| WO84/03520 | Sep., 1984 | WO | .
|
| WO89/06700 | Jul., 1989 | WO.
| |
| WO90/01068 | Feb., 1990 | WO.
| |
| WO91/17442 | Nov., 1991 | WO.
| |
Other References
English Language Abstract for Japanese Patent JP-A-02 131 599, 21 May 1990,
from Database WPIL, week 9026, Application Number (AN) =90-198034, Derwent
Publications Ltd., London, Great Britain.
R. Saiki et al., Enzymatic Amplification of .beta.-Globin Genomic Sequences
and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia (1985)
Science, 230:1350-1354.
G. Krupp and Soll, Simplified in vitro Synthesis of Mutated RNA Molecules
(1987) Febs Letters 212:271-275.
D.Y. Kwoh et al., Transcription-Based Amplification System and Detection of
Amplified Human Immunodeficiency Virus Type 1 With A Bead-Based Sandwich
Hybridization Format (1989) Proc. Natl. Acad. Sci. 86:1173-1177.
D.A. Melton et al., Efficient in vitro Synthesis of Biologically Active RNA
and RNA Hybridization Probes from Plasmids Containing a Bacteriophage SP6
Prmoter (1984) Nucleic Acids Res. 12(18):7035-7056.
M. Chamberlain et al., Bacteriophage DNA-Dependent RNA Polymerases "The
Enzymes," Boyer Ph.D., ed. (1982) 15:87-108.
C. Martin et al., Kinetic Analysis of T7 Polymerase-Promter Interactions
With Small Synthetic Promoters (1987) Biochemistry 26:2690-2696.
J. Oakley et al., Structure of a Promoter For T7 RNA Polymerase, (1977)
Proc. Natl. Natl. Sci., 74(10):4266-4270.
J. Dunn et al., Complete Nucleotide Sequence of Bacteriophage T7 DNA and
the Locations of T7 Genetic Elements (1983) J. Molec. Biol. 166:477-535.
J.F. Milligan et al., Oligoribonucleotide Synthesis Using T7 RNA Polymerase
and Synthetic DNA Template (1987) Nucleic Acids Res. 15(21):8783-8799.
P. Lizardi, et al., Exponential Amplification of Recombinant-RNA
Hybridization Probes (1988) Bio/Technology 6:1197-1202.
H. Lomell et al. Quantitative Assays Based on the Use of Replicatable
Hybridization Probes (1989) Clin. Chem. 35(9):1826-1831.
B. Chu et al., Synthesis of an Amplifiable Reporter RNA for Bioassays
(1986) Nuc. Acids Res. 35(9):1826-1831.
Saito et al., Proc. Natl. Acad. Sci. (1980) 77:3917-3921.
Osterman et al., Biochemistry (1981) 20:4884-4892.
Petty, Nucleic Acids Res. (1988) 16(17):8738.
|
Primary Examiner: Arthur; Lisa B.
Attorney, Agent or Firm: Goldman; Kenneth M., Blackburn; Robert P.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This application is a continuation of application Ser. No. 07/639,560,
filed Jan. 10, 1991, now abandoned, which is a continuation-in-part of
U.S. patent application Ser. No. 07/463,022, filed 10 Jan. 1990; now
abandoned, which applications are herein incorporated by reference in its
entirety and to which we claim priority under section 25 U.S.C. .sctn.120.
Claims
I claim:
1. An isolated polydeoxynucleotide construct for use as a signal amplifier
in hybridization assays to detect a target, the construct comprising three
domains:
(a) a first domain (A) which is single-stranded and has a nucleotide
sequence complementary to a target sequence;
(b) a second domain (B) which is double-stranded and capable of functioning
as a promoter for a DNA-dependent RNA polymerase enzyme activity; and
(c) a third domain (C) which is either single- or double-stranded and
adjacent to said second domain, such that said third domain is capable of
functioning as a template for the promoter activity of said second domain,
wherein the domains of the construct are oriented A-B-C or B-C-A, and
further wherein the third domain capable of functioning as a template
consists of a nucleotide sequence not found in the target.
2. The polydeoxynucleotide construct of claim 1 in which said DNA-dependent
RNA polymerase activity is derived from the bacteriophage T7.
3. The polydeoxynucleotide construct of claim 1 in which said DNA-dependent
RNA polymerase activity is derived from the bacteriophage T3.
4. The polydeoxynucleotide construct of claim 1 in which said DNA-dependent
RNA polymerase activity is derived from the bacteriophage SP6.
5. The polydeoxynucleotide construct of claim 1 in which said first domain
A is at least 10 and no more than 40 nucleotides long.
6. The polydeoxynucleotide construct of claim 1 in which said first domain
A is at least 15 and no more than 30 nucleotides long.
7. The polydeoxynucleotide construct of claim 5 in which said second domain
B is at least 12 and no more than 40 nucleotides long.
8. The polydeoxynucleotide construct of claim 5 in which said second domain
B is at least 17 and no more than 30 nucleotides long.
9. The polydeoxynucleotide construct of claim 7 in which said third domain
C is at least 30 and no more than 10000 nucleotides long.
10. The polydeoxynucleotide construct of claim 7 in which said third domain
C is at least 40 and no more than 80 nucleotides long.
11. The polydeoxynucleotide construct of claim 7 in which said third domain
C is at least 2 Kb and no more than 10 Kb in length.
12. The polydeoxynucleotide construct of claim 7 in which said third domain
C is at least 3 Kb and no more than 4 Kb in length.
13. The polydeoxynucleotide construct of claim 7 in which said third domain
C comprises the genomic DNA of Hepatitis B virus.
14. The polydeoxynucleotide construct of claim 2 in which the sequence for
said second domain B comprises the sequence:
' -TAA TAC GAC TCA CTA TA-3'
3'-ATT ATG CTG AGT GAT AT-5' (SEQ ID NO: 118).
15. The polydeoxynucleotide construct of claim 2 in which the nucleotide
sequence of said second domain B is:
5'-CTG GCT TAT CGA AAT TAA TAC GAC TCA CTA TA-3'
3'-GAC CGA ATA GCT TTA ATT ATG CTG AGT GAT AT-5' (SEQ ID NO: 119).
16. The polydeoxynucleotide construct of claim 1 in which the 5' residue of
the upper strand of the nucleotide sequence for said third domain C when
double-stranded, is adjacent to said second domain B, and is a guanosine
residue.
17. The polydeoxynucleotide construct of claim 1 in which the sequence for
said third domain C is:
__________________________________________________________________________
5'-GGG
AGA TGT GGT TGT CGT ACT TAG CGA AAT ACT GTC CGA
GTC G-3'
3'-CCC
TCT ACA CCA ACA GCA TGA ATC GCT TTA TGA CAG GCT
CAG C-5' (SEQ. ID NO. 120).
__________________________________________________________________________
18. The polydeoxynucleotide construct of claim 17 in which the 3' end of
the upper strand of the DNA nucleotide sequence of said second domain B is
attached to the 5' end of the upper strand of said nucleotide sequence of
domain C.
19. The polydeoxynucleotide construct of claim 1 in which a transcript
produced from of said third domain C has two subdomains:
(a) a first subdomain, c.sub.1, which is capable of hybridizing to an
oligonucleotide capture linker, said capture linker being capable of
hybridizing to a polynucleotide immobilized on a solid substrate; and
(b) a second subdomain, c.sub.2, which is capable of binding to an
oligonucleotide amplifier linker, said amplifier linker capable of binding
to a quantifiable probe.
20. The polydeoxynucleotide construct of claim 1 in which said second and
third domains, B and C, are present in multiple repeating units.
21. A method to detect and quantify an oligonucleotide analyte by
amplifying a biological signal in a nucleic acid hybridization assay
comprising:
(i) immobilizing said analyte, directly or indirectly, on a solid
substrate; and hybridizing the polydeoxynucleotide construct of claim 1,
directly or indirectly to the analyte;
(ii) removing unhybridized polydeoxynucleotide constructs;
(iii) transcribing multiple copies of RNA oligomers which are complementary
to the template sequence, c', of said third domain, C, of said
polydeoxynucleotide construct via a DNA-dependent RNA polymerase activity;
and
(iv) detecting the amount of RNA transcripts formed in step (iii).
22. The nucleic acid hybridization assay of claim 21 in which said second
domain, B, is derived from the DNA-dependent RNA polymerase of the
bacteriophage T7.
23. The nucleic acid hybridization assay of claim 21 in which said second
domain, B, is derived from the DNA-dependent RNA polymerase of the
bacteriophage T3.
24. The nucleic acid hybridization assay of claim 21 in which said second
domain, B, is derived from the DNA-dependent RNA polymerase of the
bacteriophage SP6.
25. The nucleic acid hybridization assay of claim 21 in which said
polydeoxynucleotide construct is hybridized directly to a single-stranded
oligonucleotide analyte.
26. The nucleic acid hybridization assay of claim 21 wherein said
polydeoxynucleotide construct is hybridized to an oligonucleotide linker,
said linker having a domain which is capable of forming stable hybrids
with the oligoucleotide analyte.
27. The nucleic acid hybridization assay of claim 21 in which:
(a) said third domain, C, of said polydeoxynucleotide construct is
transcribed in the presence of labeled ribonucleotide triphosphates;
(b) the transcripts of said third domain have a first subdomain, c.sub.1,
complementary to a oligonucleotide capture probe which is immobilized on a
solid substrate; and
(c) said transcripts are immobilized and quantified.
28. The nucleic acid hybridization assay of claim 21 in which:
(a) said third domain, C, of said polydeoxynucleotide construct is
transcribed in the presence of biotinylated ribonucleotides triphosphates;
(b) said transcript of said third domain has a first subdomain, c.sub.2,
which is complementary to a labeled oligonucleotide probe; and
(c) said transcripts are immobilized upon an avidinylated solid substrate;
and
(d) said transcripts are quantified.
29. The nucleic acid hybridization assay of claim 21 in which:
(a) said third domain, C, of said polydeoxynucleotide construct is
transcribed in the presence of both labeled and biotinylated
ribonucleotide triphosphates;
(b) said transcripts are immobilized upon an avidinylated solid substrate;
and
(c) said transcripts are quantified.
30. The nucleic acid hybridization assay of claim 21 in which:
(a) the transcript of said third domain, C, of said polydeoxynucleotide
construct has two subdomains:
(i) a first subdomain, c.sub.1, which is complementary to an
oligonucleotide capture probe, said probe being immobilized on a solid
substrate; and
(ii) a second subdomain, c.sub.2, which is complementary to a labeled
oligonucleotide probe;
(b) said transcript is hybridized to said capture probe;
(c) said labeled probe is hybridized to said transcripts; and,
(d) said transcripts are quantified.
31. The nucleic acid hybridization assay of claim 21 in which:
(a) the transcript of said third domain, C, of said polydeoxynucleotide
construct has two subdomains:
(i) a first subdomain, c.sub.1, which is complementary to a transcript
capture probe, said transcript capture probe being capable of hybridizing
to an oligonucleotide which has been immobilized on a solid substrate; and
(ii) a second subdomain, c.sub.2, which is complementary to a linker probe,
said linker probe being capable of hybridizing to a labeling
oligonucleotide;
(b) said transcript is hybridized to said transcript capture probe and to
said linker probe to form a transcript sandwich;
(c) said transcript sandwich is hybridized to an oligonucleotide
immobilized on a solid substrate;
(d) said immobilized sandwich is hybridized to a labelling oligonucleotide;
and
(e) said transcripts are quantified.
32. The nucleic acid hybridization assay of claim 21 in which:
(a) the transcript of said third domain, C, of said polydeoxynucleotide
construct has two subdomains:
(i) a first subdomain, c.sub.1, which is complementary to a transcript
capture probe, said transcript capture probe being capable of hybridizing
to an oligonucleotide which has been immobilized on a solid substrate; and
(ii) a second subdomain, c.sub.2, which is complementary to an amplifier
linker probe, said linker probe being capable of hybridizing to an
amplifier probe;
(b) said transcript is hybridized to said transcript capture probe and to
said amplifier linker probe to form a transcript sandwich;
(c) said transcript sandwich is hybridized to an oligonucleotide
immobilized on a solid substrate;
(d) said immobilized sandwich is hybridized to an amplifier probe;
(e) said amplifier probe is hybridized to a labeling oligonucleotide; and
(e) said transcripts are quantified.
33. The nucleic acid hybridization assay of claim 32 in which the RNA
transcript is transcribed from DNA of Hepatitis B Virus.
34. The nucleic acid hybridization assay of claim 21 in which said second
and third domains, B and C, of said polydeoxynucleotide construct are
present in multiple repeating units.
35. The method of claim 21 used to detect the presence of N. gonorrhoeae in
a biological sample in which the analyte comprises a DNA or RNA segment of
N. gonorrhoeae.
36. The method of claim 21 used to detect the presence of Hepatitis B virus
in a biological sample in which the analyte comprises a DNA or RNA segment
of Hepatitis B virus.
37. The method of claim 21 used to detect the presence of bacteria
containing the beta-Lactamase TEM-1 gene, in a biological sample in which
the analyte comprises a DNA or RNA segment of the beta-Lactamase TEM-1
gene.
38. The method of claim 21 used to detect the presence of Chlamydia in a
biological sample in which the analyte comprises a DNA or RNA segment of
Chlamydia.
39. The method of claim 21 used to detect the presence of bacteria
containing the tet M determinant in a biological sample in which the
analyte comprises a DNA or RNA segment of the tet M determinant.
40. The method of claim 21 used to detect the presence of human
immunodeficiency virus (HIV) in a biological sample in which the analyte
comprises a DNA or RNA segment of HIV.
41. The method of claim 21 used to detect the presence of hepatitis C virus
(HCV) in a biological sample in which the analyte comprises a DNA or RNA
segment of HCV.
42. A method to detect and quantify a ligand receptor by amplifying a
biological signal in a nucleic acid hybridization assay comprising the
following steps:
(a) immobilizing said ligand receptor directly or indirectly on a solid
phase;
(b) binding to the ligand receptor a ligand specific for said receptor,
said ligand being coupled to an oligonucleotide complementary to the first
domain of the construct of claim 1,
(c) removing unhybridized ligand;
(d) hybridizing the polydeoxynucleotide construct of claim 1 with the
oligonucleotide coupled to the bound ligand;
(e) removing unhybridized polydeoxynucleotide constructs;
(f) transcribing multiple copies of RNA oligomers which are complementary
to the template sequence, c', of the third domain, C, of said
polydeoxynucleotide construct via a DNA-dependent RNA polymerase activity;
and
(e) quantifying the RNA transcripts.
43. The hybridization assay of claim 42 in which the ligand receptor is an
antigen and the ligand is an antibody which immunologically reacts with
the antigen.
Description
BACKGROUND OF THE INVENTION
1. Technical Field
This invention pertains to the detection and quantification of biomolecules
by hybridization assay, and pertains more particularly to hybridization
assays wherein reporter molecules are used for signal amplification.
2. Background
Nucleic acid hybridizations are now commonly used in genetic research,
biomedical research and clinical diagnostics to detect and quantify
particular nucleotide sequences which are present in heterogeneous
mixtures of DNA, RNA, and/or other materials. In the basic nucleic acid
hybridization assay, single-stranded analyte nucleic acid (either DNA or
RNA) is hybridized, directly or indirectly, to a labeled nucleic acid
probe, and the duplexes containing label are quantified. Both radioactive
and nonradioactive labels have been used.
The basic assay lacks sensitivity. When the analyte is present in low copy
number or dilute concentration the signal cannot be distinguished from the
background noise. Variations of the basic scheme have been developed to
facilitate separation of the target duplexes from extraneous material
and/or to amplify the analyte sequences in order to facilitate detection,
but these variations have suffered generally from complex and time
consuming procedures, high background, low sensitivity, and difficulty in
quantification. A primary object of the present invention is to provide an
amplifier for use in hybridization assays that provides a highly
reproducible gain in signal, a highly reproducible signal-to-noise ratio,
is itself quantifiable and reproducible, and is capable of combining
specifically with an analyte present at low concentration and with a
"universal" reporter moiety to form a stable complex.
Commonly owned U.S. Pat. No. 4,868,105, issued 19 Sep. 1989, the disclosure
of which is hereby incorporated by reference, describes a solution phase
hybridization sandwich assay in which the analyte nucleic acid is
hybridized to a "labeling probe" and to a "capturing probe". The
probe-analyte complex is coupled by hybridization to a solid-support. This
permits the analyte oligonucleotide to be removed from solution as a solid
phase complex, thereby concentrating the analyte, facilitating its
separation from other reagents, and enhancing its subsequent detection.
PCT Application 84/03520 and EPA 124221 describe a DNA hybridization assay
in which: (1) analyte is annealed to a single-stranded DNA probe that has
a tail that is complementary to an enzyme-labeled oligonucleotide, and (2)
the resulting tailed duplex is hybridized to an enzyme-labeled
oligonucleotide. The Enzo Biochem "Bio-Bridge" labeling system appears to
be similar to the system described in these two patent applications. The
"Bio-Bridge" system uses terminal deoxynucleotide transferase to add
unmodified 3'-poly T-tails to a DNA probe. The poly T-tailed probe is
hybridized to the target DNA sequence and then to a biotin-modified poly
A.
EPA 204510 describes a DNA hybridization assay in which analyte DNA is
contacted with a probe that has a tail, such as a poly dT-tail, and an
amplifier strand that has a sequence, e.g., a poly dA sequence, that
hybridizes to the tail of the probe and is capable of binding a plurality
of labeled strands.
The main problem with these prior hybridization assays is that they lack
sufficient specificity and/or signal to be useful for detecting very low
levels of analyte.
Another commonly owned EP Application No. 88309697.6 (publication No.
0317077), filed 17 Oct. 1988, the disclosure of which is hereby
incorporated by reference, describes linear and branched oligonucleotides
which can be used as a signal amplifiers in hybridization assays. Here the
amplifier oligomer has two domains--a first domain which is complementary
to a target sequence (either the analyte per se or a "linker probe") and a
second domain, present in repeating units, complementary to a labeled
reporting sequence. The multiplication of reporting sequences per target
sequence provides for the amplification of the signal.
Another approach has been to use nucleic acid polymerases to amplify target
sequences. For example, the so-called polymerase chain reaction (PCR),
uses repeated cycles of DNA primed, DNA-directed DNA polymerase synthesis
to amplify sequences of interest (Saiki, R. K., et al., Science (1986)
230:1350-1354). The amplified target is then detected using the basic
hybridization assay protocol.
RNA polymerases have also been used to amplify target sequences (Krupp, G.,
and Soll, D. FEBS Letters (1987) 212:271-275). This approach has been
incorporated into a hybridization format that involves production of a
double-stranded copy of the target sequence, insertion of a RNA polymerase
promoter sequence, transcription of the copy and detection by
hybridization assay (Kwoh, D. Y., et al., Proc. Natl. Acad. Sci. U.S.A.
(1989) 86:1173-1177). Since DNA-directed RNA polymerases produce up to 103
copies of RNA per copy of DNA template, fewer cycles of amplification are
required. Bacteriophage DNA-dependent RNA polymerases (e.g., T3, T7, SP6)
have previously been employed for the preparation in vitro of specific RNA
sequences from cloned or synthetic oligonucleotide templates and are well
understood (Melton, D. A., et al., Nucleic Acids Res. (1984)
12:7035-7056); Chamberlin, M. and Ryan, T., (1982) in "The Enzymes,"
Boyer, P. D., ed., 15:87-108; Martin, C. T., and Coleman, J. E.,
Biochemistry (1987) 26:2690-2696). These polymerases are highly promoter
specific. DNA sequences from 17 T7 promoters are known and a consensus
sequence has been deduced (Oakley, J. L., and Coleman, J. E., Proc. Natl.
Acad. Sci. U.S.A. (1977) 74:4266-4270; Dunn, J. J., and Studier, F. W., J.
Molec. Biol. (1983) 166:477-535). It is also known that to retain
polymerase activity, only the promoter region must be double-stranded
(Milligan, J. F., et al., Nucleic Acids Res. (1987) 15:8783-8799).
Finally, RNA-directed RNA polymerase has also been used to detect target
sequences (Lizardi, P. M., et al., Bio/technology (1988) 6:1197-1202;
Lomeli, H., et al., Clin. Chem. (1989) 35:1826-1831). In this system an
RNA probe is prepared by coupling RNA complementary to the target sequence
with RNA (MDV-1) (U.S. Pat. No. 4,786,600) which serves as an exclusive
template for the bacteriophage Q-beta (Q) replicase. First, the target is
immobilized on a solid substrate, then the RNA probe is hybridized to the
target and finally the probe is eluted. Subsequent addition of
Q-beta-polymerase to the probe generates multiple copies of the
template/target RNA. In a related assay, MDV-1 RNA was first bound to
biotin, then coupled to an avidinylated target, and subsequently assayed
as described above (Chu, B. C. F., et al., Nucleic Acids Res. (1986)
14:5591-5603).
The use of Q-beta-replicase in hybridization assays has four major
disadvantages:
1) Q-beta-replicase is typically contaminated with MDV-1 RNA. Consequently,
this system has very high background (poor signal-to-noise ratio) when the
reporter sequence is the MDV-1 sequence itself;
2) The probe is RNA. RNA is highly sensitive to degradation from the RNAase
activity which is ubiquitous in crude cellular preparations, and from the
alkaline conditions required to denature double-stranded DNA targets;
3) Due to the secondary structure of MDV-1 RNA there is considerable
nonspecific binding in hybridization assays, thus significantly lowering
the sensitivity of the assay and precluding accurate quantification; and,
4) The amount of signal (the RNA product of Q-beta-replicase) varies with
the log of the number of probes originally bound to the target. Thus, this
assay can only detect order-of-magnitude differences between the
concentrations of analyte in various samples.
The invention disclosed herein has several advantages over the
Q-beta-replicase method. First, the probe is DNA rather than RNA. Second,
the assay has very high signal to noise ratio and very high sensitivity.
Third, since the signal is amplified rather than the target, the oligomer
which is actually measured will always have the same sequence and size,
thereby enabling the standardization and optimization of assay conditions
(in addition, most of the biological reagents can be used universally
thereby further simplifying and standardizing the assay). Finally, the
target can be easily and accurately quantified.
SUMMARY OF THE INVENTION
One aspect of the invention is a polydeoxynucleotide construct (template
probe) for use as a signal amplifier in hybridization assays. The template
probe is comprised of three domains as depicted in FIG. 1A:
(i) a first domain (A) which is single-stranded and has a nucleotide
sequence (a') complementary to a target sequence (a) (FIG. 2A) the target
sequence comprising a domain either within the analyte sequence or within
the sequence of an oligonucleotide which also contains a sequence domain
complementary to the analyte sequence;
(ii) a second domain (B) which is double-stranded and capable of function
as a promoter for a DNA-dependent RNA polymerase enzyme activity; and
(iii) a third domain (C) which is either single- or double-stranded and
adjacent to the second domain, such that the third domain is capable of
functioning as a template (c') for the promoter activity of the second
domain (FIG. 2B).
A second aspect of the invention is a method of amplifying the biological
signal used to detect and quantify an oligonucleotide, or other
biomolecular analyte, in a hybridization assay comprising the following
steps:
(i) immobilizing the analyte, directly or indirectly, on a solid substrate;
and hybridizing the polydeoxynucleotide template probe described supra,
directly or indirectly, to the analyte;
(ii) next removing the unhybridized template probe;
(iii) next transcribing (via a DNA-dependent RNA polymerase activity)
multiple copies of RNA oligomers (c) which are complementary to the
template sequence (c') of domain C of the amplifier; and
(iv) finally quantifying the RNA transcripts.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic representation of a monomeric template probe.
Capital letters designate domains, and lower case letters designate
strands within a domain. A primed letter designates a lower strand (read
3'- to 5'-, left to right) The a' sequence is complementary to a target
sequence. The B domain is the promoter for a RNA polymerase. The c'
sequence is the template for the RNA polymerase. The probe is synthesized
as a single strand. The AAAAAAA represents the poly-A linker added to
allow for self-annealing.
FIG. 1B is the DNA sequence of one embodiment of the template probe. The
promoter domain, B, consists of the consensus sequence of the
bacteriophage T7 promoter (SEQ ID NO: 116) plus 15 additional residues 5'
to the promoter sequence.
FIG. 1C is a schematic representation of a multimeric template probe in
which the double-stranded regions are self-annealing.
FIG. 2A is a schematic representation of a sandwich hybridization assay
system which incorporates the template probe. The analyte is indirectly
immobilized upon a solid substrate by hybridization to the analyte capture
probe and indirectly joined to the template probe via the template linker
probe. "w'" represents the sequence of a region of the immobilized
polynucleotide which is complementary to a region (w) of the analyte
capture probe. "y" represents the sequence of a region of the analyte
capture probe which is complementary to a region (y') of the analyte. "x'"
represents the sequence of a region of the analyte which is complementary
to a region (x) of the template linker probe. "a" represents a sequence of
the template linker probe which is complementary to a', the sequence of
the A domain of the template probe.
FIG. 2B is a schematic representation of the use of RNA polymerase
transcripts as reporter molecules in a hybridization assay. After
hybridization of the analyte and template probe, an RNA polymerase is
added and multiple RNA transcripts complementary to the template sequences
(c) are produced. These sequences have two sub-domains: c.sub.1 which is
complementary to a capture probe immobilized upon a solid substrate; and
c.sub.2 which is complementary to a labeling probe. This allows for
indirect immobilization of the label and easy quantification of the
hybridization assay signal. "*" designates the incorporated label which
may be radioactive, chemiluminescent, fluorescent or enzymatic.
FIG. 2C is the DNA sequence of a transcript of the C domain, as well as the
sequence of the transcript capture probe (C.sub.1 '), and the labeling
probe (C.sub.2 '). X represents the N.sup.4 methyl deoxycytidine
derivative, [(6-aminocaproyl)-2-aminoethyl]-5-methyl-2'-deoxycytidine,
used to couple the capture probe to the solid phase.
FIG. 3A depicts the preparation of pII template probe.
FIG. 3B depicts the preparation of pII.sub.L template probe.
FIG. 3C depicts the preparation of pII.sub.R template probe.
FIG. 4 depicts the domain A oligomer of Example 9 and the DNA sequences of
oligo N, oligo S and oligo H.
FIG. 5 depicts a protocol for a nucleic acid assay utilizing the T7
template probe and also utilizing an amplifier probe to further increase
sensitivity and amplification of the signal.
FIG. 6 is a graph showing the relative sensitivity and signal amplification
of various template probes in assays for the presence of HIV in human
serum.
DETAILED DESCRIPTION OF THE INVENTION
A "biological signal" is a biochemically transmitted indicium of the
occurrence of an event or presence of a specific molecule.
"DNA-dependent RNA polymerase" is an enzyme which facilitates the
polymerization of RNA of specific sequence from a complementary DNA
template.
A "domain" is a particular region of a polynucleotide characterized by its
function.
An "immunological reaction" is the specific recognition and binding of an
antibody to its corresponding epitope.
A "polydeoxynucleotide" is a polymeric DNA molecule. A "polynucleotide" is
a polymeric DNA or RNA molecule.
A "promoter" is the site on a polydeoxynucleotide to which a RNA polymerase
enzyme binds preparatory to initiating transcription.
"RNA-dependent RNA polymerase" is an enzyme which facilitates the
polymerization of RNA of specific sequence from a complementary RNA
template.
"Transcription" is a process, mediated by an enzyme, by which RNA is formed
corresponding to a complementary polynucleotide template.
The "upper strand" of a double-stranded DNA molecule is the strand whose
5'-end is on the left as the sequence is read from left to right. The
sequence of this strand is always presented above the sequence for its
complementary "lower strand" which is read 3'- to 5'-, left to right.
MODES FOR CARRYING OUT THE INVENTION
1. Template Probe
In one aspect of this invention a DNA probe (referred to as a "template
probe") containing three functional domains has been designed in order to
enhance the signal in hybridization assays.
The first domain ("A" in FIG. 1A), has a sequence (a') usually 10 to 40
nucleotides in length, preferably 15 to 30 nucleotides, is single-stranded
and is designed to hybridize to a complementary target sequence (a). In
order to achieve hybrid stability, this domain will normally have a GC
content in the range of 40% to 60%. The target sequence may subsist within
the overall sequence of the polynucleotide to be assayed (referred to as
the analyte) or it may subsist within an oligonucleotide linker which also
has homology to the analyte. In a preferred embodiment, the analyte will
be immobilized upon a solid substrate to facilitate subsequent washing
procedures. This immobilization may be direct (e.g., polynucleotide
preparations containing the analyte might be bound to a nitrocellulose
filter) or indirect (e.g., a linker might be immobilized on the filter and
the analyte subsequently hybridized to the linker).
The second domain (B), usually 10 to 40 basepairs in length, preferably 20
to 35 nucleotides, more preferably 30 to 35 nucleotides, is
double-stranded and functions as a DNA-directed RNA polymerase promoter.
This promoter is usually derived from the promoter sequence of a
bacteriophage, preferably any of the phage T3, T7, or SP6, more preferably
from bacteriophage T7. This class of RNA polymerases is highly promoter
specific. The T7 promoter is probably the best characterized. DNA
sequences from 17 T7 promoters are known and a consensus sequence had been
deduced: (SEQ ID NO: 116) (Oakley and Coleman; Dunn and Studier).
Sequences 3' to the promoter on the complementary strand (the c' segment,
whose 3' end is adjacent to the 5' end of the b' segment) serve as the
template for transcription and the transcription of many template sequence
variations can be accommodated. Only the promoter region itself must be
double-stranded (Milligan et al.).
Additional sequences may be added at the 5' end of the promoter. For
example, in a preferred embodiment, the B region consists of the consensus
sequence of the T7 promoter plus additional bases 5' to the consensus
sequence which are identical to the sequence of the pT7 plasmids
(available from US Biochemicals) up to the PvuII restriction site (FIG.
1B). These sequences may or may not have an effect on transcription.
The third domain (C) is directly 3' to the second domain and the c' strand
of this domain serves as the template for the domain B promoter. Domain C
may be as small as 30 nucleotides in length, or as long as 10 Kb. In a
preferred embodiment the domain is 40 to 45 bases. In another preferred
embodiment the domain is 3.4 Kb and is substantially similar to the
genomic DNA of Hepatitis B virus. This domain may be either single- or
double-stranded, and the 3' end of the c' template strand (directly
adjacent to the promoter) usually is a cytosine residue.
The proper 5' to 3' relation of the promoter (B domain) to the template (C
domain) is necessary for proper transcription of the template. The
promoter is directly 5' to the template and the template is read 3' to 5'.
However, it will be appreciated by those skilled in the art that the
orientation of the B/C domains to the A domain is not critical. Thus
template probes constructed as domains A-B-C, or as B-C-A will produce the
same transcript and therefore may be constructed in either form.
The RNA transcription product (c) of the C domain functions as a reporter
molecule for the presence and quantity of analyte. Signal amplification
occurs because each template produces 10.sup.1 to 10.sup.4 transcripts.
The sequence of this domain may be designed with a random sequence,
evaluated by computer analysis to minimize the possibility of
cross-reaction with other probes in the system, or alternatively, may be a
known sequence which has been specifically chosen.
Further amplification can be achieved by designing the template probe with
multimeric promoter/template (B/C) domains (FIG. 1C). These multimeric
units may be either in a linear array or branched molecules. For further
details concerning the technology for the production and application of
such multimers in hybridization assays, see EPA publication No. 0317077.
In a multimeric template probe the total number or repeating B/C units will
usually be in the range of 2 to 200, more usually 5 to 20. The B/C units
of the multimer may be covalently linked directly to each other through
phosphodiester bonds or through interposed linking agents such as nucleic
acid, amino acid, carbohydrate or polyol bridges, or through other
cross-linking agents that are capable of cross-linking nucleic acid
strands. The site(s) of linkage may be at the ends of the unit (in either
normal 3'-5' orientation or randomly) and/or at one or more internal
nucleotides in the strand. In linear multimers the individual units are
linked end-to-end to form a linear polymer. In branched multimers three or
more oligonucleotide units emanate from a point of origin to form a
branched structure. The point of origin may be another oligonucleotide
unit or a multifunctional molecule to which at least three units can be
covalently bound. The multimer may be totally linear, totally branched, or
a combination of linear and branched portions. Preferably there will be at
least two branch points in the multimer, more preferably at least three.
The multimer may include one or more segments of double-stranded
sequences.
Template probes may be prepared by cloning, enzymatic assembly, chemical
cross-linking techniques, direct chemical synthesis or a combination
thereof. When prepared by cloning, nucleic acid sequences that encode the
entire probe or fragments thereof can be made in single- or
double-stranded form by conventional cloning procedures. When made in
double-stranded form, the probe is denatured to provide single strands.
Template probes may also be cloned in single-stranded form using
conventional single-stranded phage vectors such as M13.
The A domain is single-stranded, the B domain is double-stranded, and the C
domain may be either single- or double-stranded. A particular domain
(e.g., B domain) can subsequently be made double-stranded by hybridization
with its complementary strand--cloned separately. Alternatively, the
entire template probe can be cloned as a single-stranded, self-annealing
polynucleotide (a' b' c' c b). In this case four to ten additional
nucleotides, preferably 5-7 nucleotides, are added to the sequence as a
spacer between c and c' to allow for proper contouring of the
double-stranded region when it is self-annealed. The spacer is usually
poly-A, but may be modified to minimize hybridization cross-reactivity
between various probes in the assay.
If multimeric probes are desired, fragments are linked enzymatically or
chemically to form the multimer. When assembled enzymatically, the
individual units are ligated with a ligase such as T4 DNA ligase. When
prepared by chemical cross-linking, the individual units may be
synthesized with one or more nucleic acids that have been derivatized to
have functional groups that provide linking sites or derivatized after the
oligonucleotide has been synthesized to provide such sites. A preferred
procedure for chemical cross-linking is to incorporate N.sup.4 -modified
cytosine bases into the nucleotide as described in the commonly owned EPA
publication No. 0225807.
When prepared by direct chemical synthesis oligonucleotides containing
derivatized nucleic acids whose functional groups are blocked are made by
conventional oligonucleotide synthesis techniques. The functional groups
are unblocked and oligonucleotide units are synthesized out from the
unblocked site(s).
2. Amplified Hybridization Assay
Another aspect of this invention employs template probes in hybridization
assays. The analyte may be any nucleotide sequence of interest--either DNA
or RNA. The analyte sequence may constitute an entire molecule or only a
portion of a molecule. The analyte may be a homogeneous polynucleotide,
present in low concentration in a prepared sample or it may be a minority
species in a heterogeneous mixture of polynucleotides. The analyte may
also be from a variety of sources, e.g., biological fluids or tissues,
food stuffs, environmental materials, etc., or it may be synthesized in
vitro.
The analyte may be prepared for the hybridization analysis by a variety of
means, e.g., proteinase K/SDS, chaotropic salts, etc. Also, it may be of
advantage to decrease the average size of the analyte by enzymatic,
physical or chemical means, e.g., restriction enzymes, sonication,
chemical degradation (e.g., metal ions), etc. The fragments may be as
small as 0.1 kb, usually being at least about 0.5 kb and may be 1 kb or
higher. Where the analyte sequence is lengthy, for example a viral genome,
several different regions of the analyte may be used as targets of an
analyte probe.
The analyte sequence is provided in single-stranded form for analysis.
Where the sequence is naturally present in single-stranded form,
denaturation will not usually be required. However, where the sequence is
present in double-stranded form, the sequence will be denatured.
Denaturation can be carried out by various techniques, such as alkali,
generally from about 0.05 to 0.2M hydroxide, formamide, chaotropic salts,
heat, or combinations thereof.
In a first step, the analyte may be immobilized directly upon a solid phase
or by sandwich hybridizations in which the analyte is bound to an
oligonucleotide that is in turn bound to a solid phase. A particularly
useful approach is a solution phase sandwich hybridization described in
commonly owned EPA publication No. 0225807.
In a sandwich hybridization assay with a capture step the template probe is
used as follows: Single-stranded analyte nucleic acid is incubated under
hybridization conditions with an excess of two single-stranded nucleic
acid probes, (1) an analyte capture probe having a first binding sequence
complementary to the analyte and a second binding sequence that is
complementary to a single-stranded oligonucleotide bound to a solid phase,
and (2) a template linker probe having a first binding sequence that is
complementary to the analyte and a second binding sequence that is
complementary to domain A of the template probe.
In a preferred embodiment, a set of analyte capture probes may be used
wherein each member of the set has a different first binding sequence
complementary to a different segment of the analyte while all members of
the set have the same second binding sequence. Similarly, a set of
template linker probes may be used wherein each member of the set has a
different first binding sequence complementary to a different segment of
the analyte, but all members of the set have the same second binding
sequence complementary to domain A of the template probe. This approach
has the advantage of enabling the simultaneous detection of closely
related variants of an analyte, e.g. the genomes of related viral strains.
By using analyte capture and template linker probes, the solid matrix and
the template probe can be used as a "universal" reagent and different
immobilized oligonucleotide matrices and template probes need not be made
for each analyte.
Usually, hybridization conditions consist of an aqueous medium,
particularly a buffered aqueous medium, which includes various additives.
These additives include the polynucleotides to be hybridized, salts (e.g.,
sodium citrate 0.017M to 0.17M and sodium chloride 0.17M to 1.7M),
nonionic or ionic detergents (0.1 to 1.0%), and carrier nucleic acids.
Nonaqueous solvents such as dimethylformamide, dimethylsulfoxide, and
formamide may also be used. The mixture is incubated for 15 to 75 minutes
at 45.degree. C. to 70.degree. C. The stringency of the hybridization is
regulated by temperature and salt concentration and may be varied
depending on the size and homology of the sequences to be hybridized. For
hybridization of sequences to bound DNA, the empiricl formula for
calculating optimum temperature under standard conditions (0.9M NaCl) is:
T(.degree.C.)=4(N.sub.G +N.sub.C)+2(N.sub.A +N.sub.T)-5.degree. C.,
where N.sub.G, N.sub.C, N.sub.A, and N.sub.T are the percentage of G, C, A,
and T bases in the sequence (Meinkoth, J., et al., Anal. Biochem. (1984)
138:267-284).
The resulting product is a three component nucleic acid complex of the two
probes hybridized to the analyte by their first binding sequences. The
second binding sequences of the template linker probe and analyte capture
probe remain as single-stranded tails as they are not complementary to the
analyte.
This complex is then added under hybridizing conditions to a solid phase
having a single-stranded oligonucleotide bound to it that is complementary
to the second binding sequence of the analyte capture probe. The resulting
product comprises the complex bound to the solid phase via the duplex
formed by the oligonucleotide bound to the solid phase and the second
binding sequence of the analyte capture probe. The solid phase with bound
complex is then separated from unbound materials and washed to remove any
residual unbound material.
The template probe is then added to the solid phase-analyte-probe complex
under hybridization conditions to permit the template probe to hybridize
to the available second binding sequences of the template linker probe of
the complex (the target sequence of the template probe). The resulting
solid phase complex is then separated from any unbound template probe and
washed.
Next, the RNA polymerase specific for the promoter region (domain B) of the
template probe is added under appropriate transcription conditions and
multiple RNA copies (c) of the C domain template (c') are produced. The
amount of transcript is proportional to the quantity of the analyte in the
initial preparation.
Transcription conditions consist of an aqueous medium, preferably a
buffered aqueous medium, with appropriate salts, usually including a
magnesium salt, a mixture of NTPs (rATP, rUTP, rGTP, rCTP), a RNA
polymerase enzyme and usually include various denaturing agents, protein
carriers, and RNAse inhibitors. Incubation is usually for 15 to 90
minutes, usually 60 minutes; and at a temperature which is optimal for the
chosen enzyme, usually 35.degree. C. to 42.degree. C., usually 37.degree.
C.
The sequence of the C domain is designed for a specific detection scheme
and several such schemes may be employed to quantify the transcripts. For
example, the transcription product (c) of the C domain may be subdivided
into 2 subdomains--c.sub.1 and c.sub.2 (FIG. 2B). Subdomain c.sub.1 is
complementary to a transcript capture probe which has been immobilized on
a solid substrate. Subdomain c.sub.2 is complementary to an amplifier
probe. After hybridization the amount of label retained is linearly
proportional to the amount of analyte present in the original sample. In a
variation of this approach, the transcripts are sandwiched with linker
probes, i.e., the transcript capture probe is in solution rather than
immobilized, and contains a second domain which is complementary to an
immobilized oligonucleotide; and subdomain c.sub.2 is complementary to an
amplifier linker probe which in turn is complementary to the amplifier
probe. This sandwiching arrangement is similar to the use of analyte
capture and template linker probes to sandwich the analyte as described
above.
In an alternate embodiment the transcript of the C domain has only a
c.sub.1 subdomain. The C domain is transcribed in the presence of labeled
ribonucleotide triphosphates and the labeled transcript is subsequently
bound to an immobilized transcript capture probe through its complementary
c.sub.1 subdomain and quantified.
In yet another embodiment the transcript of the C domain has only a c.sub.2
subdomain. The C domain is transcribed in the presence of biotinylated
ribonucleoside triphosphates and the transcripts is captured on avidin
beads. The transcript is then annealed to an amplifier probe through its
complementary c.sub.2 subdomain and quantified.
Several other methods of labeling and detecting the transcript of the
amplifying probe are possible, including the simultaneous use of labeled
ribonucleotides and avidin/biotin coupling, and will be obvious to those
skilled in the art.
Capture, Linker and Amplifier Probes
The first binding sequences of the analyte capture probe and template
linker probe are complementary to the analyte sequence. Similarly, the
first binding sequences of the transcript capture and amplifier linker
probes are complementary to the reporter transcripts. Each first binding
sequence is at least 12 nucleotides (nt), usually at least 25 nt, more
usually at least 30 nt, and not more than about 150, usually not more than
about 75, preferably not more than about 50 nt. They will normally be
chosen to bind to different sequences of the analyte. The first binding
sequences may be selected based on a variety of considerations. Depending
upon the nature of the analyte, one may be interested in a consensus
sequence, a sequence associated with polymorphisms, a particular phenotype
or genotype, a particular strain, or the like.
The second binding sequences of the analyte capture probe and template
linker probe are selected to be complementary, respectively, to the
oligonucleotide attached to the solid phase and to an oligonucleotide unit
of the template probe and so as to not be encountered by endogenous
sequences in the sample/analyte. The second binding sequence may be
contiguous to the first binding sequence or be spaced therefrom by an
intermediate noncomplementary sequence. The probes may include other
noncomplementary sequences if desired. These noncomplementary sequences
must not hinder the binding of the binding sequences or cause nonspecific
binding to occur.
The capture probes and linker probes may be prepared by conventional
oligonucleotide synthesis procedures or by cloning.
It will be appreciated that the binding sequences need not have perfect
complementarity to provide homoduplexes. In many situations,
heteroduplexes will suffice where fewer than about 10% of the bases are
mismatches, ignoring loops of five or more numbers. Accordingly, as used
herein the term "complementary" intends a degree of complementarity
sufficient to provide a stable and specific duplex structure.
The solid phase that is used in the assay may be particulate or be the
solid wall surface of any of a variety of containers, e.g., centrifugal
tubes, columns, microtiter plate wells, filters, tubing, etc. Preferably,
particles will be employed of a size in the range of about 0.4 to 200
microns, more usually from about 0.8 to 4.0 microns. The particles may be
any convenient material, such as latex, or glass. The oligonucleotide that
is complementary to the second binding sequence of the analyte capture
probe may be stably attached to the solid surface through functional
groups by known procedures.
It will be appreciated that one can replace the second binding sequence of
the capture probe and the oligonucleotide attached to the solid phase with
an appropriate ligand-receptor pair that will form a stable bond joining
the solid phase to the first binding sequence of the capture probe.
Examples of such pairs are biotin/avidin, thyroxine/thyroxine-binding
globulin, antigen/antibody, carbohydrate/lectin, and the like.
The amplifier probes will include a sequence complementary to the C.sub.2
subdomain of the transcripts of the template probe, or to a subdomain of
an amplifier linker probe. The amplifier probe is capable of hybridizing
to one or more labels or labeling probes which directly or indirectly
provide for a detectable signal. The labels may be incorporated in
individual residues of the complementary sequence or may be present as a
terminal domain or terminal tail having a plurality of labels. Various
means for providing labels bound to the sequence have been reported in the
literature. See, for example, Urdea et al., Nucl. Acids Res. (1988) 4937;
Leary et al., Proc. Natl. Acad. Sci. USA (1983) 80:4045; Renz and Kurz,
Nucl. Acids Res. (1984) 12:3435; Richardson and Gumport, Nucl. Acids Res.
(1983) 11:6167; Smith et al., Nucl. Acids Res. (1985) 13:2399; Meinkoth
and Wahl, Anal. Biochem. (1984) 138:267. The labels may be bound either
covalently or noncovalently to the complementary sequence. Labels which
may be employed include radionuclides, fluorescers, chemiluminescers,
dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,
enzyme subunits, metal ions, and the like. Illustrative specific labels
include fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone,
luminol, NADPH, galactosidase, horseradish peroxidase, alkaline
phosphatase, etc. See Urdea et al. for a comparison of nonradioisotopic
labeling methods.
The labeling probes can be conveniently prepared by chemical synthesis such
as that described in commonly owned copending application Ser. No.
945,876. By providing for a terminal group which has a convenient
functionality, various labels may be joined through the functionality.
Thus, one can provide for a carboxy, thiol, amine, hydrazine or other
functionality to which the various labels may be joined without
detrimentally affecting duplex formation with the sequence. As already
indicated, one can have a molecule with a plurality of labels joined to
the sequence complementary to the labeling sequence. Alternatively, one
may have a ligand bound to the labeling sequence and use a labeled
receptor for binding to the ligand to provide the labeled analyte complex.
The ratio of analyte capture probe and template linker probe to anticipated
moles of analyte will each be at least stoichiometric and preferably in
excess. This ratio is preferably at least about 1.5:1, and more preferably
at least 2:1. It will normally be in the range of 2:1 to 10,000:1.
Concentrations of each of the probes will generally range from about
10.sup.-9 to 10.sup.-6 M, with sample nucleic acid concentrations varying
from 10.sup.-21 to 10.sup.-12 M. The hybridization steps of the assay will
generally take from about 10 minutes to 2 hours, frequently being
completed in about 1 hour. Hybridization can be carried out at a mildly
elevated temperature, generally in the range from about 20.degree. C. to
80.degree. C., more usually from about 35.degree. C. to 70.degree. C.,
particularly 65.degree. C. Additional conditions for the hybridization
reaction are described infra.
The procedure used in the separation steps of the assay will vary depending
upon the nature of the solid phase. For particles, centrifugation or
filtration will provide for separation of the particles, discarding the
supernatant or isolating the supernatant. Where the particles are assayed,
the particles will be washed thoroughly, usually from one to five times,
with an appropriate buffered medium containing detergent, e.g., PBS with
SDS. When the separation means is a wall or support, the supernatant may
be isolated or discarded and the wall washed in the same manner as
indicated for the particles.
Depending upon the nature of the label, various techniques can be employed
for detecting the presence of the label. For fluorescers, a large number
of different fluorometers are available. With enzymes, either a
chemiluminescent, fluorescent or a colored product can be provided and
determined fluorometrically, spectrophotometrically or visually. The
various labels which have been employed in immunoassays and the techniques
applicable to immunoassays can be employed with the subject assays.
In a hybridization assay in which the analyte nucleic acid is bound
directly to a solid phase, such as a "dot blot" assay, the template probe
is hybridized directly to the bound analyte. In these instances, the A
domain of the template probe is complementary to a sequence of the
analyte.
The template probe may also be used in other assays such as direct,
indirect, and sandwich immunoassays and assays for ligand receptors, for
instance cell surface receptors. In these instances, rather than a label,
the reagent that plays the role of the labeled antibody, or other ligand
which binds to the analyte (antigen or ligand receptor), has an attached
oligonucleotide that is complementary to a', the sequence of the A domain
of the template probe. For instance, in a sandwich immunoassay for an
antigen analyte, the analyte sample is incubated with a solid phase to
which is bound a first antibody to the antigen. Unbound sample is removed
from the solid phase and a second antibody to the antigen, which is
coupled to an oligonucleotide complementary to a', is reacted with the
bound complex to form a three-membered complex. Following removal of
excess second antibody the template probe is then hybridized to the
complex via the oligonucleotide bound to the second antibody. Excess
template probe is removed and RNA polymerase is added as described supra.
Finally, the transcription product is quantified as described.
In an alternative embodiment, the template probe may be synthesized without
an A domain and coupled directly to the ligand receptor or antibody by
means of avidin/biotin or an equivalent stably bonding pair as described
previously. The template probe may also be covalently attached to the
ligand receptor by means of the chemical synthesis described in copending
application Ser. No. 06/945,876, now U.S. Pat. No. 5,093,232, issued Mar.
3, 1992.
Kits for carrying out amplified nucleic acid hybridization assays according
to the invention will comprise in packaged combination the following
reagents: the template probe; the appropriate DNA-directed RNA polymerase;
an appropriate labeling probe; a solid phase that is capable of binding to
the analyte; optionally an analyte capture probe if the assay format is
one in which the analyte is bound to the solid phase through an
intermediate oligonucleotide or other ligand; and optionally a template
linker probe if the assay format is one in which the template probe is not
hybridized directly to the analyte. Similarly, these kits may also contain
transcript capture probes amplifier probes and amplifier linker probes.
These reagents will typically be in separate containers in the kit. The
kit may also include a denaturation reagent for denaturing the analyte,
hybridization buffers, wash solutions, negative and positive controls and
written instructions for carrying out the assay.
EXAMPLES
Example 1
A. T7 Template Probe
The probe was designed as shown in FIG. 1A. The a' sequence of the A domain
is shown in FIG. 1B and is complementary to the a region of the template
linker probe depicted in FIG. 2A. The sequence of the promoter domain, B,
(shown in FIG. 1B) contained the consensus T7 promoter sequence plus 15
additional bases 5' to the promoter and identical to the sequence of the
pT7 plasmid (available from US Biochemicals) up to the PvuII restriction
site. The additional 15 basepairs may be extraneous; however, they were
incorporated since the initial experiments conducted with template probes
that had been cloned into the pT7 vector proved successful. Thus, even
template probes made by chemical synthesis have retained this plasmid
portion. The C domain was designed as a random sequence. It was evaluated
by computer analysis to minimize potential hybridization cross-reactivity
with other probes in the system.
B. T3 Template Probe
The probe is designed as in Example 1A, above, except that the consensus
sequence for the DNA-directed RNA polymerase promoter sequence of
bacteriophage T3 (SEQ ID NO: 2) TATTAACCCTCACTAAA is substituted for the
consensus sequence for the T7 promoter TAATACGACTCACTATA (SEQ ID NO: 1).
C. SP6 Template Probe
The probe is designed as in Example 1A, above, except that the consensus
sequence for the DNA-directed RNA polymerase promoter sequence of
bacteriophage SP6 ATTTAGGTGACACTATA (SEQ ID NO: 3) is substituted for the
consensus sequence for the T7 promoter and the first six 5' nucleotides of
domain C are 5'-GAAGGG-3' rather than 5'-GGGAGA-3, as is the case in
Example 1A.
Example 2
Hybridization Assay for the Pilin Gene DNA of Neisseria gonorrhoeae Using a
Microtiter Dish Assay Procedure and the T7 RNA Polymerase
A. Standard Analyte DNA
The N. gonorrhoeae strain 31707 from the Neisseria Reference Laboratory
(Seattle, Wash.) was used. DNA was prepared from this strain, as well as
from several nonpathogenic commensal strains of Neisseria used as
controls, by the addition of a proteinase K/SDS solution as described in
Urdea et al. (Gene (1987) 61:253).
B. Oligonucleotide Bound to Solid Support (FIG. 2A)
A microtiter dish assay procedure was employed. Microtiter dishes were
prepared as follows. Two types of microtiter dish wells were prepared: (1)
N wells for sample work-up and negative controls, and (2) S wells for
capture of the probe-analyte complex from samples and positive controls.
N wells were produced as follows: 300 .mu.l of HM buffer (0.1% SDS,
4.times.SSC, 1 mg/ml sonicated salmon sperm DNA, 1 mg/ml poly A, 10 mg/ml
BSA) was added to Immulon II Remov-a-wells (Dynatech Inc.). The well
strips were covered and left standing at room temperature for 1 hour. The
HM buffer was removed by aspiration and the wells were washed 3 times with
400 .mu.l of 1.times.PBS. The strips were covered with plastic wrap and
stored at 4.degree. C. until used.
S wells were prepared from the Immulon II strips as follows. To each well,
200 .mu.l of a 200 .mu.g/ml solution of poly-phenylalanyl-lysine (Sigma
Chemical Inc.) in water. The covered strips were left at room temperature
for 30 min to 2 hr, then washed as above.
Next, a 21 base oligomer, XCACCACTTTCTCCAAAGAAG (SEQ ID NO: 4), where X
represents the N4-(6-aminocaproyl-2-aminoethyl) derivative of 5-methyl
cytidine, was synthesized according to the method of Warner et al. (DNA
(1984) 3:401) and purified as described by Urdea et al., supra. The
N4-modified cytosine base facilitates the chemical cross-linking of the
oligonucleotide as described in commonly owned EPA Publication No. 0225807
and Urdea, M. S., et al., Nucl. Acids Res. (1988) 16:4937-4956.
A 10 OD sample of the synthesized oligonucleotide in 60 .mu.l of
1.times.PBS was treated with 140 .mu.l of dimethyformamide containing 10
mg of ethylene glycol bis (succinimidylsuccinate) (Pierce Chemical Inc.).
The mixture was vortexed and incubated in the dark at room temperature.
After 15 min, the solution was passed over a Sephadex.RTM. G-25 column
(PD-10 from Pharmacia), previously equilibrated with 30 ml of 1.times.PBS.
The void volume of the column was diluted to a final volume of 35 ml with
1.times.PBS. To each well, a 50 .mu.l aliquot of the oligonucleotide
solution was added. After covering with plastic wrap, the wells were
incubated at room temperature in the dark for 30 min to overnight. The
wells were washed with 1.times.PBS, then coated with HM buffer, washed,
and stored as above.
C. Analyte capture Probes (FIG. 2A)
A set of 3 single-stranded oligomers each having a varying 30 base long
portion complementary to a specific sequence of the pilin gene and a
constant 20 base long 5'-portion complementary to the oligonucleotide
bound to the solid phase was synthesized by the automated phosphoramidite
procedures described in Warner et al., supra, and purified by the method
of Sanchez-Pescador and Urdea, supra. The sequences complementary to the
pilin gene were based on the N. gonorrhoeae pilin sequence described by
Bergstrom, S., et al. (PNAS USA (1986) 83:3890-3894). The 5' portions of
the probes were complementary to segments of the pilin sequence and were
as follows:
__________________________________________________________________________
Probe Designation
5'-Sequence
__________________________________________________________________________
GCP-XT1-4 GAT GTG GCG GGC GCG CGT TCA AAG GCT TCG
(SEQ ID NO: 5)
GCP-XT1-8 GAG GCT GTA GTT TCC GTT TAT ACA ATT TCT
(SEQ ID NO: 6)
GCP-XT1-12
GCC AAG CCA TTT TAC CAA GAC GCC TGT CGG
(SEQ ID NO: 7)
__________________________________________________________________________
The 3'-portion of each analyte capture probe was constructed to be
complementary to the sequence of the oligonucleotide attached to the solid
support described infra.
D. Template Linker Probes (FIG. 2A)
A set of 12 single-stranded oligomers each consisting of a varying 30 base
long portion complementary to a specific sequence of the pilin gene and a
constant 20 base long 3'-portion complementary to the template probe (FIG.
2A) were synthesized by the procedures of Warner et al., supra and
purified according to Sanchez-Pescador and Urdea, supra.
The 5' portions of the probes were complementary to segments of the pilin
sequence and were as follows:
______________________________________
Probe Designation 5'-Sequence
______________________________________
GCP-LLA2C-1 (SEQ ID NO: 8)
GCP-LLA2C-2 (SEQ ID NO: 9)
GCP-LLA2C-3 (SEQ ID NO: 10)
GCP-LLA2C-5 (SEQ ID NO: 11)
GCP-LLA2C-6 (SEQ ID NO: 12)
GCP-LLA2C-7 (SEQ ID NO: 13)
GCP-LLA2C-9 (SEQ ID NO: 14)
GCP-LLA2C-10 (SEQ ID NO: 15)
GCP-LLA2C-11 (SEQ ID NO: 16)
GCP-LLA2C-13 (SEQ ID NO: 17)
GCP-LLA2C-14 (SEQ ID NO: 18)
GCP-LLA2C-15 (SEQ ID NO: 19)
______________________________________
The 3'-portion of each template linker probe was constructed to be
complementary to the sequence of the A domain of the template probe.
E. Labeled Oligomer (FIG. 2B)
An 18 base oligomer, XGGTCCTAGCCTGACAGC (SEQ ID NO: 20), where X is defined
as above, was synthesized as described, and combined with alkaline
phosphatase (AP) as follows: Calf intestinal AP (3mg in buffer;
immunoassay grade, Boehringer-Mannheim) was placed in a Centricon 30
Microconcentrator. Approximately 2 ml of 0.1M sodium borate, pH 9.5, was
then added and the device was spun at 3500 rpm until a final volume of 40
.mu.l was obtained. The alkylamino oligonucleotide was then activated with
p-phenylene diisothiocyanate (DITC; Pierce Chemicals) in 95:5 (v/v)
dimethylformamide: 0.1M sodium borate, pH 9.3, extracted with n-butanol,
and combined with the protein. The final product was stored at 4.degree.
C. See Urdea et al. (Nuc. Acids Res. (1988) 16:4937).
F. Microtiter Dish Procedure
For duplicate analyses, 20 .mu.l of each sample was placed into 2 N wells,
then treated with 25 .mu.l of proteinase K/SDS solution. The wells were
covered with a Linbro-Titertek microtiter plate sealer, gently agitated,
and incubated at 65.degree. C. for 30 min in a water bath. The analyte
capture and template linker probe sets in a 1M NaOH were added in 10 .mu.l
to each well. After sealing, the samples were incubated for 10-30 min at
65.degree. C. to 72.degree. C. as above. The solutions were neutralized
with 26 .mu.l 0.38M acetic acid (or 0.76M 3-[N-morpholino]propane sulfonic
acid (MOPS), free acid), 12.3.times.SSC, then incubated for an additional
15-30 min covered at 65.degree. C. From each N well, 40 .mu.l of sample
was transferred to a new S well containing the solid supported capture
probe. The wells were sealed and set at 65.degree. C. for 1 hour. Each
well was then washed 2 times by aspiration with 0.1% SDS, 0.1.times.SSC.
See Folberg et al. (Molec. and Cell. Probes (1989) 3:59).
The template probe was subsequently annealed to the complex by incubation
of 100 fmoles of template probe in 40 .mu.l of 4.times.SSC with 100
.mu.g/ml poly A at 55.degree. C. for 1 hr followed by two washes with
0.1.times.SSC, 0.1% SDS and two washes with 0.1.times.SSC.
Transcription of domain C was effected by incubating the complex in 20
.mu.l of a solution containing 40 mM Tris HCl (pH 8), 20 mM MgCl.sub.2, 10
mM NaCl, 1 mM Spermidine, 10 mM Dithiothreitol, 0.15 mg/ml Bovine Serum
Albumin, 1.25 mM each of rATP, rCTP, rGTP, rUTP, 1600 units/ml RNasin, and
2000 units/ml T7 RNA polymerase. This mixture was incubated at 37.degree.
C. for 1 hour. Transcription was terminated by addition of 20 .mu.l of a
solution containing 8.times.SSC, and 0.2% SDS and the entire mixture was
transferred to new wells containing an immobilized capture probe with
c.sub.1 ' sequences. Capture of the domain C transcripts (FIG. 2B) was
effected by incubation at 55.degree. C. for 1 hour followed by two washes
with 0.1.times.SSC, 0.1% SDS.
The domain C transcripts were then labeled by addition of 50 fmol of
enzyme-labeled probe (c.sub.2 ') in 40 .mu.l of 4.times.SSC, 100 .mu.g/ml
poly A for 15 min. at 55.degree. C. Finally, the complex was washed twice
with 0.1.times.SSC, 0.1% SDS, followed by two washes with 0.1.times.SSC.
For AP detection, an enzyme-triggered dioxetane-based reaction (Schapp et
al. Tet. Lett. (1987) 28:1159-1162) and U.S. Pat. No. 4,857,652),
available from Lumigen Inc., was employed. The detection procedure was as
follows. For the labeling step 40 .mu.l HM buffer with the AP probe was
added to each well and the wells were incubated at 55.degree. C. for 15
min. The supernatant was removed and the wells were washed 2.times. with
380 .mu.l of 0.1.times.SSC and 0.1% SDS. The wells were then washed
2.times. with 380 .mu.l of 0.1.times.SSC to remove any remaining SDS. 20
.mu.l of 3.3.times.10.sup.-4 M dioxetane reagent in CTAB buffer was added
to each well. The wells were tapped lightly so that the reagent would fall
to the bottom and gently swirled to distribute the reagent evenly over the
bottom. The wells were covered with the microtiter plate sealer and
incubated in a 37.degree. C. oven for one hour. The wells were then read
with a luminometer.
Results
Tests were carried out on N. gonorrhoeae bacterial cells (strain 31707) as
well as nonpathogenic Neisseria controls according to the protocol of
Example 2, above. Results are presented as a signal to noise ratio (S/N)
representing the value of the sample versus the value of the control. Cell
number was determined by cell viability. For comparison, tests were also
carried out on the same samples using a branched 5-site comb-type
amplification multimer described in copending application U.S. Ser. No.
109,282.
__________________________________________________________________________
T7 Transcription Multimeric
Cell Assay Assay
Number
Trial 1 Trial 2 Trial 1
Trial 2
__________________________________________________________________________
8.3 .times. 10.sup.5
186.76 .+-. 25.39
139.93 .+-. 44.87
90.94 .+-. 48.69
82.75 .+-. 16.55
8.3 .times. 10.sup.4
18.35 .+-. 3.60
8.35 .+-. 4.20
9.94 .+-. 8.71
14.56 .+-. 0.47
8.3 .times. 10.sup.3
2.43 .+-. 0.37
1.55 .+-. 0.37
2.58 .+-. 1.40
2.55 .+-. 0.42
__________________________________________________________________________
Example 3
Hybridization Assay Using the T3 RNA Polymerase
A hybridization assay is employed using the same protocol as in Example 2,
supra, except that domain B of the template probe contains the sequences
for the T3 RNA polymerase promoter rather than the T7 promoter. The
sequence of the T3 promoter has been previously disclosed by Brown, J. E.,
et al. (Nucleic Acids Res. (1986) 14:3521-3526). The T3 promoter sequence
is TCA CTA AAG GGA GA-3' (SEQ ID NO: 21) and replaces the T7 promoter
sequence TAA TAC GAC TCA CTA TAG GGA GA (SEQ ID NO: 22).
Example 4
Hybridization Assay Using the SP6 RNA Polymerase
A hybridization assay is employed using the same protocol as in Example 2,
supra, except that domain B of the template probe contains the sequence
for the SP6 RNA polymerase promoter rather than the T7 promoter. The
sequence of the SP6 promoter has been previously disclosed by Brown et
al., supra. The SP6 promoter sequence is (SEQ ID NO: 23) and replaces the
T7 promoter sequence (SEQ ID NO: 22).
Example 5
Hybridization Assay for Hepatitis B Virus (HBV) DNA Using the Microtiter
Dish Assay Procedure and T7 RNA Polymerase
DNA extracts of serum or plasma samples of patients potentially infected
with hepatitis B virus (HBV) are prepared as described in copending U.S.
application Ser. No. 109,282 and are used as analyte as described in
Example 2.
A set of single-stranded template linker probes, each having a varying 30
base long portion complementary to a specific sequence of the constant ds
region of the HBV genome and a constant 20 base long 3'-portion
complementary to the template probe used in Example 2 is synthesized by
the procedures described in Example 2. The sequences of these probes are
presented in Table 1 below.
TABLE 1
______________________________________
Template Linker Probes for HBV
Probe Designation Sequence
______________________________________
:HBV.LLA2C.70 (SEQ ID NO: 24)
:HBV.LLA2C.69 (SEQ ID NO: 25)
:HBV.LLA2C.68 (SEQ ID NO: 26)
:HBV.LLA2C.67 (SEQ ID NO: 27)
:HBV.LLA2C.66 (SEQ ID NO: 28)
:HBV.LLA2C.65 (SEQ ID NO: 29)
:HBV.LLA2C.59 (SEQ ID NO: 30)
:HBV.LLA2C.58 (SEQ ID NO: 31)
:HBV.LLA2C.57 (SEQ ID NO: 32)
:HBV.LLA2C.56 (SEQ ID NO: 33)
:HBV.LLA2C.55 (SEQ ID NO: 34)
:HBV.LLA2C.54 (SEQ ID NO: 35)
:HBV.LLA2C.53 (SEQ ID NO: 36)
:HBV.LLA2C.52 (SEQ ID NO: 37)
:HBV.LLA2C.51 (SEQ ID NO: 38)
______________________________________
A set of single-stranded analyte capture probes, each having a varying 30
base-long portion complementary to a specific sequence of the constant ds
region of the HBV genome and a constant 20 base long 3'-portion
complementary to the oligonucleotide bound to a microtiter dish as
described in Example 2 is synthesized as described in Example 2. The
sequences of these probes are presented in Table 2 below.
TABLE 2
______________________________________
Analyte Capture Probes for HBV
Probe Designation Sequence
______________________________________
:HBV.XT1.64 (SEQ ID NO: 39)
:HBV.XT1.63 (SEQ ID NO: 40)
:HBV.XT1.62 (SEQ ID NO: 41)
:HBV.XT1.61 (SEQ ID NO: 42)
:HBV.XT1.60 (SEQ ID NO: 43)
______________________________________
All other methods and reagents are the same as Example 2.
Example 6
Hybridization Assay for TEM-1 beta-Lactamase DNA in N. gonorrhoeae Using
the Microtiter Dish Assay Procedure and T7 RNA Polymerase
Molecular analyses have revealed that the penicillin resistance observed in
N. gonorrhoeae is mostly due to the presence of a TEM-1 beta-Lactamase
gene in a nonconjugative plasmid of 3-7 M. daltons. (This plasmid is
homologous to those found in H. ducreyi, H. parainfluenzae, and
occasionally H. influenzae.) A hybridization assay is thus developed to
detect TEM-1 DNA in N. gonorrhoeae (or the other aforementioned bacteria
carrying homologous plasmids) for the purpose of determining penicillin
resistance.
The 7.3 Kb N. gonorrhoeae plasmid carrying the TEM-1 gene has been obtained
and a segment containing 80% of the TEM-1 gene was sequenced as described
in commonly owned EPA Publication No. 0317077. Analyte capture and
template linker probes are synthesized and purified as described in
Example 2, supra. The 5'-portion of the template linker probes are
complementary to sequences of the coding region of the gene; whereas the
5'-portions of the analyte capture probes are complementary to adjoining
sequences of the plasmid. Alternatively, probes are also prepared in which
the 5'-portions of both sets are directed to the TEM-1 gene.
In all other respects the hybridization assay procedure and reagents are
the same as described in Example 2.
This TEM-1 assay is a powerful clinical tool that will enable medical
personnel to identify penicillin-resistant infection and optimize a
treatment regime by choosing an appropriate antibiotic therapy.
Example 7
Hybridization Assay for Chlamydia trachomatis DNA Using the Microtiter Dish
Assay procedure and T7 Polymerase
Template linker and analyte capture probes are prepared using the same
strategy as described in Example 2 and designed to hybridize to the
Chlamydia pCHL2 plasmid described by Palmer and Falkow (Plasmid (1986)
16:52-62). Each probe of the set is a 50 mer in which the first 30
5'-residues are complementary to pCHL2 sequences and the remaining
3'-residues are the system-specific analyte capture and template linker
sequences described in Example 2. The pCHL2 sequences used to design these
probes are disclosed in commonly owned EPA Publication No. 0317077.
In all other respects the hybridization assay procedure and reagents are
the same as describe in Example 2.
Example 8
Hybridization Assay for tet M Determinant in N. gonorrhoeae
N. gonorrhoeae strains resistant to high levels of tetracycline, exhibiting
minimum inhibitory concentration values above 16 g/ml, have been found to
have acquired the tet M determinant in a 24.5 Md conjugative plasmid
(Cannon, J. G., et al., Annual Review of Microbiology (1984) 38:111;
Morse, S. A., et al., Antimicrob. Agents Chemother. (1986) 30:664). A
hybridization assay is thus developed to detect tet M DNA in N.
gonorrhoeae (or the other aforementioned bacteria carrying homologous
plasmids) for the purpose of determining tetracycline resistance.
Ten .mu.l of tetracycline resistant N. gonorrhoeae (TRNG) cells suspended
in either GC broth or skimmed milk are mixed with 12.5 .mu.l of lysis
solution (2mg/ml proteinase K in 10 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA,
1% SDS, pH 8.0) in a clear Immulon II well (Dynatech), and incubated at
65.degree. C. for 20 min.
Analyte capture and template linker probes are synthesized and purified as
described in Example 2, supra, except they are designed to hybridize to
the tet M structural gene. The sequences of the probes are based on the
tet M gene sequence from the streptococcal conjugative shuttle transposon
Tn 1545 described in Martin, P., et al., Nuc. Acids Res. (1986) 14:7047.
In all other respects the hybridization assay procedure and reagents are
the same as described in Example 2.
Example 9
Comparison of Template Probes with Various Numbers of Base Pairs Between
the A and B Domain in Assays for the Presence of Human Immunodeficiency
Virus (HIV) DNA in Human Plasma
Various template probes were prepared, each having the same functional
domains, A, B, and C, but with different numbers of base pairs separating
the A domain and the T7 promoter.
The general strategy was to prepare DNA containing the T7 polymerase
promoter operably linked to the 5' end of the Hepatitis B viral genome
(HBV)--about 3.4 Kb in length. Thus, the HBV genome acts as domain C and
functions as the template for subsequent transcription while the resulting
HBV-specific RNA functions as reporter transcripts. This DNA fragment was
cloned in plasmid pGEM3Z (commercially available from Promega, Inc.--see
FIG. 3A). Next, a partially single stranded oligomer, corresponding to the
A domain of the template probes, and a short (18 nucleotide) double
stranded spacer region with a cohesive end was also prepared (FIG. 4). The
oligomer was then ligated to the promoter/HBV DNA fragment (B/C domain)
which had been isolated from the plasmid by restriction endonuclease
digestion and subsequent purification. The size of the fragment varied
depending on the restriction endonuclease used to linearize the plasmid
(FIGS. 3A-C).
A. pII Template Probe
1. T7 Promoter (Domain B) and HBV (Domain C)
A DNA segment comprised of the T7 consensus sequence, oriented directly 5'
to linearized HBV genomic DNA, was inserted in the EcoR1 site of pGEM3Z
(pGEM3Z-HBV). After cloning, the plasmid was isolated and relinearized by
digestion with Nde I (FIG. 3A).
2. The Oligomer
A partially double stranded oligomer was synthesized which comprised domain
A and a short double stranded domain terminating in a cohesive end
complementary to the cohesive end created by Nde I digestion. This
oligomer is designated oligo N. Oligo N is comprised of two DNA strands.
The sequence of strand X is (SEQ ID NO: 44). The sequence of strand Y is
(SEQ ID NO: 45) AT-5'. Strands X and Y were annealed creating a single
stranded domain A at the 3'-end of strand Y and an AT cohesive end at the
5'-end of strand Y.
3. Ligation
Oligo N and Nde I linearized pGEM3Z-HBV were ligated, creating a
oligonucleotide with oligo N at both ends of the molecule (FIG. 3A). The
molecule was trimmed with Hind III thereby creating the template probe
designated pII (FIG. 3A). The distance between the T7 promoter and domain
A is 200 base pairs.
B. pII.sub.L Template Probe
pII.sub.L template probe was synthesized as described in A, supra, except
that pGEM3Z-HBV was linearized with Sca I, thereby creating a longer
spacer region between the T7 promoter of domain B and domain A (FIG. 3B).
In this case, the oligomer is designated oligo S and consists of strands X
and Y'. Oligo S differs from oligo N in that there is no cohesive end.
Strand X is the same as strand X in oligo N, but strand Y' lacks the
5'-TA. Strands X and Y' were annealed creating a single stranded domain A
at the 3'-end of strand Y' and a blunt end at the 5'-end of strand Y. The
linearized plasmid and oligo S were blunt-end ligated. The distance
between the T7 promoter and domain A is 900 base pairs.
C. pII.sub.R Template Probe
pII.sub.R template probe was synthesized as described in A, supra, except
that pGEM3Z-HBV was linearized with Hind III (FIG. 3C). The oligomer is
designated oligo H and consists of strands X and Y". Oligo H differs form
oligo N in that the cohesive end is complementary to the cohesive end
generated by HindD III digestion. Strand X is the same as strand X in
oligo N, but strand Y" has the sequence (SEQ ID NO: 47). Strands X and Y"
were annealed creating a single stranded domain A at the 3'-end of strand
Y" and an TCGA cohesive end at the 5'-end of strand Y". Oligo H and Hind
III-linearized pGEM3Z-HBV were ligated, creating a oligonucleotide with
oligo H at both ends of the molecule (FIG. 3C). The molecule was trimmed
with Nde I rather than Hind III, thereby creating the template probe
designated pII.sub.R. This probe differs from pII and pII.sub.L in that
the sequence of domains is B-C-A. The distance between T7 promoter and
domain A is 3200 base pairs.
D. HIV-specific Capture and Linker Probes
The assays described below (see FIG. 5) are similar to the assay described
in Example 2 above. Since HIV is the analyte it was necessary to create
analyte capture probes and template linker probes with subdomains
homologous to the HIV genome. The sequences of these novel linker probes
are provided below. The 5'-portion of each probe is complementary to a
portion of the HIV genome while the 3'-portion is complementary to an
immobilized oligonuleotide capture sequence (in the case of capture
probes) or to the A domain of the T7 template probe (in the case of the
template linker probes).
TABLE 3
______________________________________
Analyte Capture Probes
Probe Designation Sequence
______________________________________
:HIV.96.1.XT1 (SEQ ID NO: 47)
:HIV.96.2.XT1 (SEQ ID NO: 48)
:HIV.97.XT1 (SEQ ID NO: 49)
:HIV.97.2.XT1 (SEQ ID NO: 50)
:HIV.53.XT1 (SEQ ID NO: 51)
:HIV.54.XT1 (SEQ ID NO: 52)
:HIV.55.XT1 (SEQ ID NO: 53)
:HIV.68.1.XT1 (SEQ ID NO: 54)
:HIV.68.2.XT1 (SEQ ID NO: 55)
:HIV.99.XT1 (SEQ ID NO: 56)
:HIV.100.XT1 (SEQ ID NO: 57)
:HIV.101.XT1 (SEQ ID NO: 58)
:HIV.102.XT1 (SEQ ID NO: 59)
______________________________________
TABLE 4
______________________________________
Template Linker Probes
Probe Designation Sequence
______________________________________
:HIV.51.LLA2C (SEQ ID NO: 60)
:HIV.52.LLA2C (SEQ ID NO: 61)
:HIV.56.LLA2C (SEQ ID NO: 62)
:HIV.57.LLA2C (SEQ ID NO: 63)
:HIV.58.1.LLA2C (SEQ ID NO: 64)
:HIV.58.2.LLA2C (SEQ ID NO: 65)
:HIV.59.1.LLA2C (SEQ ID NO: 66)
:HIV.59.2.LLA2C (SEQ ID NO: 67)
:HIV.60.LLA2C (SEQ ID NO: 68)
:HIV.62.LLA2C (SEQ ID NO: 69)
:HIV.63.LLA2C (SEQ ID NO: 70)
:HIV.64.1.LLA2C (SEQ ID NO: 71)
:HIV.64.2.LLA2C (SEQ ID NO: 72)
:HIV.65.LLA2C (SEQ ID NO: 73)
:HIV.98.LLA2C (SEQ ID NO: 74)
:HIV.66.LLA2C (SEQ ID NO: 75)
:HIV.67.LLA2C (SEQ ID NO: 76)
:HIV.70.LLA2C (SEQ ID NO: 77)
:HIV.71.LLA2C (SEQ ID NO: 78)
:HIV.72.LLA2C (SEQ ID NO: 79)
:HIV.73.LLA2C (SEQ ID NO: 80)
:HIV.69.LLA2C (SEQ ID NO: 81)
:HIV.74.LLA2C (SEQ ID NO: 82)
:HIV.75.LLA2C (SEQ ID NO: 83)
:HIV.76.LLA2C (SEQ ID NO: 84)
:HIV.77.LLA2C (SEQ ID NO: 85)
:HIV.78.1.LLA2C (SEQ ID NO: 86)
:HIV.78.2.LLA2C (SEQ ID NO: 87)
:HIV.79.LLA2C (SEQ ID NO: 88)
:HIV.80.LLA2C (SEQ ID NO: 89)
:HIV.81.LLA2C (SEQ ID NO: 90)
:HIV.82.LLA2C (SEQ ID NO: 91)
:HIV.83.LLA2C (SEQ ID NO: 92)
:HIV.84.LLA2C (SEQ ID NO: 93)
:HIV.85.LLA2C (SEQ ID NO: 94)
:HIV.86.LLA2C (SEQ ID NO: 95)
:HIV.87.LLA2C (SEQ ID NO: 96)
:HIV.88.LLA2C (SEQ ID NO: 97)
:HIV.89.LLA2C (SEQ ID NO: 98)
:HIV.90.LLA2C (SEQ ID NO: 99)
:HIV.91.LLA2C (SEQ ID NO: 100)
:HIV.92.LLA2C (SEQ ID NO: 101)
:HIV.93.LLA2C (SEQ ID NO: 102)
:HIV.94.LLA2C (SEQ ID NO: 103)
:HIV.95.1.LLA2C (SEQ ID NO: 104)
:HIV.95.2.LLA2C (SEQ ID NO: 105)
:HIV.103.LLA2C (SEQ ID NO: 106)
______________________________________
D. Comparison of the pII Template Probes in the Assay of HIV in Human
Plasma
Samples of normal human plasma with varying amounts of a synthetic HIV
target sequence were prepared as described in Example 2. 10 .mu.l of
plasma were added to 12.5 .mu.l of extraction buffer (10 mM Tris pH 8.0,
150 mM NaCl, 10 mM EDTA, 1% SDS 40.mu.g/ml sonicated salmon sperm DNA, and
2mg/ml proteinase K) and incubated in wells of a microtiter dish at
65.degree. C. for 30 minutes. The wells were first prepared by binding a
single stranded oligonucleotide with a defined sequence to the solid
substrate as described in Example 2. 5 .mu.l of 1N NaOH with 12.5 fmoles
of HIV capture and linker probes/well (described above) were added and the
mixture was further incubated at 65.degree. C. for 30 minutes. Next 13
.mu.l of MOPS-SSC (0.77M 3-[N-morpholino]propanesulfonic acid, 1.845M
NaCl, 0.185M Na Citrate) were added and the mixture further incubated at
65.degree. C. for 2 hours.
Wells were then washed 2 times with wash buffer A (0.1.times.SSC, 0.1%
SDS). Following the washes, 30 fmoles of T7 template probe in 40 .mu.l
horse hyb mix (50% horse serum, 0.6M NaCl, 0.06M Na Citrate, 0.1% SDS)
were added and the mixture incubated at 55.degree. C. for 1 hour. (The
horse hybridization mix was prepared as follows: for ten ml--504 .mu.l
water (treated with diethyl pyrocarbonate, DEPC), 336 .mu.l 10% SDS, 60
.mu.l 1M Tris HCl (pH8), 100 .mu.l of 25 mg/ml proteinase K, 5 ml horse
serum, incubated 65.degree. C. for 2 hours, add 1 ml water (DEPC treated)
and 2 ml 20.times.SSC).
The wells were then washed 2 times with wash buffer A and 2 times with wash
buffer B (0.1.times. SSC). Next 40 .mu.l of transcription mix (40 mM
Tris-HCl pH 8, 20 mM MgCl.sub.2, 80 units T7 polymerase (New England
Biolabs), 10 mM DTT, 0.15 mg/ml BSA, 1.25 mM each of ATP, UTP, GTP, and
CTP, 1600 units/ml RNAsin) were added and the mixture incubated in a 37
degree oven for 1.5 hours.
New wells were again prepared by binding single stranded oligonucleotide
with a defined sequence to the solid substrate (described in Example 2).
To the new well was added 12.5 .mu.l of extraction buffer (2 mg/ml
proteinase K), 5 .mu.l of proteinase K/SDS treated human serum, 15 .mu.l
20.times. SSC, 5 .mu.l 10% SDS containing 12.5 fmoles of transcript
capture and amplifier linker probes, and 12.5 .mu.l of the mixture from
the first well which contains the newly formed RNA transcripts. This
mixture was incubated 65.degree. C. for 2 hours.
Note that, in contrast to the protocol of Example 2, transcript capture and
amplifier linker probes are used to sandwich the RNA transcript (FIG. 5).
These probes serve as bridges between the transcript and the immobilized
nucleotide on the one hand, and between the transcript and the amplifier
probe (to be added) on the other hand.
The wells are next washed 2 times with wash buffer A. Forty .mu.l of horse
hybridization mix containing 100 fmoles of comb-like amplifier probe (as
in Example 2) added to each well and incubated at 55.degree. C. for 15
minutes. Wells were then washed 2.times. with wash buffer A. Forty .mu.l
of horse hybridization mix containing 100 fmoles of alkaline phosphatase
probe is added to each well and incubated at 55.degree. C. for 15 minutes.
(The horse hybridization mixture is pretreated to remove residual RNAse
activity according to the preparation protocol described above except
that, after the 65 degree incubation, the solution is cooled and 60 .mu.l
of 100 mM phenyl-methyl sulfonyl fluoride (PMSF) is added to inactivate
the proteinase K, and the mixture is further incubated at 37.degree. C.
for 1 hour).
The samples were assayed by alkaline phosphatase detection as described in
Example 2 above. The wells were washed twice with wash buffer A, twice
with wash buffer B. 20 .mu.l of dioxetane reagent were added and incubated
at 37.degree. C. for 30 minutes and the wells were then read in a
luminometer.
E. Results
Panels of various amounts of cloned HIV DNA added to samples of human serum
were prepared and assayed as described above. Each panel was assayed using
one of the three PII T7-template probes described above. The sensitivity
and amplification are shown in FIG. 6. As a control standard, a panel of
HIV DNA was assayed using only the comb-like signal amplification probe
assay described in E.P. Pub. 0317077. The use of the T7 template probe
provides an approximate 30 fold increase in sensitivity and an approximate
50 fold increase in amplification relative to the non-polymerase standard.
Example 10
Hybridization Assay for Hepatitis C Virus (HCV) DNA Using the Microtiter
Dish Assay Procedure and T7 RNA Polymerase
Assays for the presence of HCV-specific DNA and RNA are preformed as
described in Example 9. Sets of template linker and analyte capture probes
are prepared as described in Example 2 and designed to hybridize to
portions of the HCV genome. These probes are disclosed in the commonly
owned PCT US90/02853, filed 18 May 1990. Each probe of the set is a 50-mer
in which the first 30 residues at the 5'-end are complementary to HCV
sequences and the remaining residues are the system-specific analyte
capture and template linker sequences described in Example 2.
In the same manner, other pathogenic bacterial, viral and parasitic strains
and antibiotic resistance-conferring genes can be screened. It will be
appreciated that the invention assay may be adapted to conduct multiple
assays for different analytes simultaneously. In one format, by changing
the label and the amplifier probe sequences for a new analyte (as well as
the analyte specific sequences in the analyte capture and template linker
probes) it is possible to detect two different analytes in the same sample
on the same solid phase. Alternatively, by synthesizing different
oligonucleotides bound to the solid support (Example 2B, supra) for each
analyte, and attaching each bound oligonucleotide sequence to different
positions on a membrane strip, it is possible to perform several different
assays simultaneously with the same label.
Modifications of the above-described modes for carrying out the invention
that are obvious to those of skill in nucleic acid chemistry, biochemical
and clinical assays and related fields are intended to be within the scope
of the following claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 120
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TAATACGACTCACTATA17
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TATTAACCCTCACTAAA17
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATTTAGGTGACACTATA17
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: modified.sub.-- base
(B) LOCATION: 1
(D) OTHER INFORMATION: /note="Represents the
N4-(6- aminocaproyl-2-aminoethyl) derivative of
5-methyl cytidine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCACCACTTTCTCCAAAGAAG21
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GATGTGGCGGGCGCGCGTTCAAAGGCTTCG30
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GAGGCTGTAGTTTCCGTTTATACAATTTCT30
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GCCAAGCCATTTTACCAAGACGCCTGTCGG30
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATACTTATGGGAAGTTTTTCCGAAATGGGA30
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCTCGACTACTAACACTAGCGATAGCAGCC30
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
AAACCGCAATCAGCGGGAAGGGCGGATGGT30
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GGAAAACCGGCTTCCAGTTTTTAGTCGGCA30
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GCTCATAATGGACTTAAGGCCGTTTACCGG30
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
TTTGTTGTGAAGACGGCCGCACCGTAGGGG30
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ACTTCAATTTTTGCCGCAGCAATGGCGGTG30
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CGAAAGTTCGCCGCATTTGTTACTAATGTT30
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GTTTTTTGAGAGGGACACCCGGTCCGCACT30
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
ATGCGCGTGGCTGCTGCTGTGGCAACGGCT30
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GTTTCTGCCGTTTCTTTAGCTGTGGTTCGT30
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CGGCAGTTGGACGGCGCTATTCCGTAGACT30
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: modified.sub.-- base
(B) LOCATION: 1
(D) OTHER INFORMATION: /note="Represents the
N4-(6- aminocaproyl-2-aminoethyl) derivative of
5-methyl cytidine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GGGTCCTAGCCTGACAGC18
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
TATTAACCCTCACTAAAGGGAGA23
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TAATACGACTCACTATAGGGAGA23
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
ATTTAGGTGACACTATAGAAGGG23
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
TAATACGACTCACTATAGGGAGA23
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
TGACTGSCGATTGGTRGAGGCAGGMGGAGGTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CTTGWYGGGRTTGAAGTCCCAATCTGGATTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GTTGCGTCAGCAAACACTTGGCASAGACCWTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
TAAGTTGGCGAGAAAGTRAAAGCCTGYTTMTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GCAGCAAARCCCAAAAGACCCACAAKWCKYTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
ATGTATACCCARAGACARAAGAAAATTGGTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
TAGAGGACAAACGGGCAACATACCTTGRTATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
GATGAGGCATAGCAGCAGGATGAAGAGGAATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
GATAAAACGCCGCAGACACATCCAGCGATATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
GGACAARTTGGAGGACARGAGGTTGGTGAGTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
TTGGAGGTTGGGGACTGCGAATTTTGGCCATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
CCACCACGAGTCTAGACTCTGYGGTATTGTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
GATTCTTGTCAACAAGAAAAACCCCGCCTGTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
CACGAGMAGGGGTCCTAGGAATCCTGATGTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
CAGGGTTTACTGTTCCKGAACTGGAGCCACTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
CTTGGCCCCCAATACCACATCATCCATATACTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
GAAAGCCAAACAGTGGGGGAAAGCCCTACGCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
CACTGAACAAATGGCACTAGTAAACTGAGCCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
GAGAAACGGRCTGAGGCCCMCTCCCATAGGCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
SCGAAAGCCCAGGAYGATGGGATGGGAATACTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
GGTCGACTAATCGGTAGC18
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
TAGCTACCGATTAGTCGACCGACACGGGTCCTATGCCT38
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
AGCTGCTACCGATTAGTCGACCGACACGGGTCCTATGCCT40
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
TTCTACTACTTTYACCCATGCRTTTAAAGCTTCTTTGGAGAAAGTGGTG49
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
TTCTATTACTTTYACCCATGCRTTCAAAGCTTCTTTGGAGAAAGTGGTG49
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
TGCTTGATGTCCCCCCACTGTGTTTAGCATCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
TGCCTGGTGTCCTCCAACTATGTTCAGCATCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
AGGTGATATGGCYTGATGTAYCATTTGCCCCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
CATGGGTATYACTTCTGGGCTRAARGCCTTCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
TTGYGGGGTGGCYCCYTCTGATAATGCTGACTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
AATTTTTRAAATTTTYCCTTCCTTTTCCATCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
AACTCTTRAAATTTTYCCTTCCTTTTCCATCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
TTACTGGTACAGTYTCAATAGGRCTAATKGCTTCTTTGGAGAAAGTGGTG50
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
TAACYYTTGGGCCATCCATYCCTGGCTTTCTTCTTTGGAGAAAGTGGTG49
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
CTTTTATTTTTTCTTCTGTCAATGGCCATCTTCTTTGGAGAAAGTGGTG49
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
AAATACTGGAGTATTGTATGGATTYTCAGCTTCTTTGGAGAAAGTGGTG49
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
TCCSCCGCTTAATACYGACGCTCTCGCACCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
TTAWATAATGATYTAAGTTCTTCTGATCCTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
ACTTCCYCTTGGTTCTCTCATYTGRCCTGGTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
TTCYTGAAGGGTACTAGTRGTTCCTGCTATTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
GATAGGTGGATTAYKTGTCATCCATSCTATTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
GATAGGTGGGTTGYKTGTCATCCATSCTATTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
ATTATCCAYCTTTTATARATTTCTCCTACTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
ATTATCCAYCTTTTATARATGTCTCCCACTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
CTATACATYCTTACTATTTTATTTAATCCCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
TTYGCATTTTGGACCARSAAGGTTTCTGTCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
CTCCCTGRCATGCTGTCATCATTTCTTCTATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
TTCAKTTGGTGTCCTTCCTTYCCACATTTCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
TTCAKTTGGTGTCCTTCCCTYCCACATCTCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
GCCARATYTTCCCTAAAAAATTAGCCTGTCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
RTCCCAKTCTGCAGCTTCCTCATTGATRGTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
ATCATTTTTGGTTTCCATYTTCYTGGCAAATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
TGTCYTACTTTGATAAAACCTCCAATTCCCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
TCTCCAYTTRGTRCTGTCTTTTTTCTTTATTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 145..1335
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
GTACTGATATCYAMTCCCTGGTGTYTCATTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
GGTGATCCTTTCCATCCCTGTGGHAGCACATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
TAAGATTTTTGTCATGCTACWYTGGAATATTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
AGAYCCTACATACAAATCATCCATGTATTGTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
TATTTTTGYTCTATGYTGYCCTATTTCTAATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
ATGYTTTTTRTCTGGTGTGGTAARTCCCCATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
ATAMCCCATCCAAAGRAATGGRGGTTCTTTTTAGGCATAGGACCCGTGGTC51
(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
TAYTAAGTCTTTTGATGGGTCATAATAYACTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
TGTTTTCAGATTTTTAAATGGYTCTTGATATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
TGTTTTCAGATTTTTATATTGYTCTTGGTATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
GYTAAYTGTTTYACATCATTAGTGTGGGCATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
GGARTYTTTCCCCATATYACTATGCTTTCTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
CCCCATCTACATAGAAVGTTTCTGCWCCTATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
TGCTTGTAAYTCAGTYTTCTGATTTGTTGTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
ATCTGGTTGTGCTTGAATRATYCCYAATGCATTAGGCATAGGACCCGTGTC51
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
ATCTACTTGTTCATTTCCTCCAATYCCTTTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
TAGCCATTGCTCTCCAATTRYTGTGATATTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
GACATTTATCACAGCTRGCTACTATTTCYTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
TATRTAKCCACTGGCTACATGRACTGCTACTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
TTTTACTGGCCATCTTCCTGCTAATTTTATTAGGCATAGGACCCGTGTC49
(2) INFORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
TACTCCTTGACTTTGGGGRTTGTAGGGAATTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
TCTYTCCCTGCCACTGTAYCCCCCAATCCCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
TAGTTTGTATGTCTGTTGCTATYATGYCTATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
TTTGAATTTTTGTRATTTGYTTTTGTARTTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:
TCCAGAGDAGYTTTGCTGGTCCTTTCCAAATTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:
TATTRTCYTGTATTACTACTGCCCCTTCACTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 145..1335
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:
TTRCTTTTCTTCTTGGCACTACTTTTATRTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:
TTRCTTTTCTTCTTGGTACTACCTTTATRTTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:
TTTTCTTTTAAAATTGTGRATGAAYACTGCTTAGGCATAGGACCCGTGTC50
(2) INFORMATION FOR SEQ ID NO:108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:
CTGGCTTATCGAAATTAATACGACTCACTATAGGGAGATGTGGTTGTCGTACTTAGCGAA60
ATACTGTCCGAGTCGAAAA79
(2) INFORMATION FOR SEQ ID NO:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:
AAACGACTCGGACAGTATTTCGCTAAGTACGACAACCACATCTCCCTATAGTGAGTCGTA60
TTAATTTCGATAAGCCAGGACACGGGTCCTATGCCTAA98
(2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: modified.sub.-- base
(B) LOCATION: 1
(D) OTHER INFORMATION: /note="Represents the
N4-(6- aminocaproyl-2-aminoethyl) derivative of
5-methyl cytidine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
CCGACTCGGACAGTATTTCGCTAAGTACGACAACCACATC40
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
GGGAGATGTGGTTGTCGTACTTAGCGAAATACTGTCCGAGTCG43
(2) INFORMATION FOR SEQ ID NO:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:
GGTCGACTAATCGGTAGC18
(2) INFORMATION FOR SEQ ID NO:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:
TAGCTACCGATTAGTCGACCGACACGGGTCCTATGCCT38
(2) INFORMATION FOR SEQ ID NO:114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:
GGTCGACTAATCGGTAGC18
(2) INFORMATION FOR SEQ ID NO:115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:
GCTACCGATTAGTCGACCGACACGGGTCCTATGCCT36
(2) INFORMATION FOR SEQ ID NO:116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:
GGTCGACTAATCGGTAGC18
(2) INFORMATION FOR SEQ ID NO:117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:
AGCTGCTACCGATTAGTCGACCGACACGGGTCCTATGCCT40
(2) INFORMATION FOR SEQ ID NO:118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) SEQUENCE DESCRIPTION: SEQ ID NO:118:
TAATACGACTCACTATA17
(2) INFORMATION FOR SEQ ID NO:119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) SEQUENCE DESCRIPTION: SEQ ID NO:119:
CTGGCTTATCGAAATTAATACGACTCACTATA32
(2) INFORMATION FOR SEQ ID NO:120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) SEQUENCE DESCRIPTION: SEQ ID NO:120:
GGGAGATGTGGTTGTCGTACTTAGCGAAATACTGTCCGAGTCG43
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